The centromere is the chromosomal domain that specifies the motile and mechanical properties of chromosomes during mitosis.Proper chromosome segregation results in aneuploidy which causes serious developmental defects, such as Down's Syndrome and may play a role in tumor progression in neoplastic diseases.The goals of this proposal are to contribute to understanding the molecular basis of chromosome movement through analysis of the function of centromere protein-A (CENP-A) in human cells. Originally defined as a scleroderma autoantigen, CENP-A has now been shown to be a novel histone H3-like protein that is localized uniquely at the centromere. This proposal will use a recently isolated full length cDNA clone for human CENP-A as a tool to dissect the biological properties of CENP-A, focusing on two main questions:1) How is CENP-A targeted specifically to the centromere and 2) What function(s) does CENP-A provide at the centromere. These questions will be pursued through 4 specific experimental aims.The location of CENP-A within the centromere will be determined by preparing antibodies to be used in immunofluorescence and immunoelectron microscopy. Preliminary experiments indicated that the mode of expression of CENP-A is critical for proper targeting to the centromere. The timing of synthesis of CENP-A in the cell cycle will be analyzed by cell synchronization methods coupled with RNA and protein blotting and metabolic labeling techniques, to determine how CENP-A synthesis is related to DNA replication and normal histone expression. To understand the structures required for specific molecular recognition and assembly of the centromere, the protein structural requirements for targeting CENP-A to centromeres will be determined using mammalian cell transfection techniques to assay targeting of chimeric molecules constructed between histone H3 and CENP-A.A role for the centromere DNA binding protein CENP-B in CENP-A localization will be investigated using co-transfection experiments. The knowledge, materials and techniques developed in these experiments will support investigation of the role of CENP-A in centromere structure and function. CENP-A structure and expression will be disrupted in human cells by expression of mutant proteins, antisense inhibition of CENP-A expression and antibody microinjection. Mitotic spindle and centromere structure will be analyzed by immunofluorescence and electron microscopy and physiological effects on mitosis and the cell cycle will be analyzed in stably transformed cells. The relationship of CENP-A to CSE4, a structurally homologous chromosome segregation gene recently identified in yeast will be determined using genetic complementation in yeast and localization assays developed for mammalian cells, with the aim of developing a shuttle system to study CENP-A function.